Neuropeptide Y (NPY) is a 36-amino acid peptide neurotransmitter found in the brain and autonomic nervous system; it is the most abundant neuropeptide in serum and plasma. NPY 1-36 has been associated with a number of physiological processes in the brain, including the regulation of energy balance, memory and learning, and epilepsy. The main effect of NPY is increased food intake and decreased physical activity. NPY 1-36 is secreted by the hypothalamus, and, in addition to increasing food intake, it increases the proportion of energy stored as fat and blocks nociceptive signals to the brain. Importantly, NPY 1-36 augments the vasoconstrictor effects of noradrenergic neurons. The full peptide is, therefore, an important diagnostic in patients suffering from hypertension, stress, and cardiovascular disease, for example.
NPY 1-36 is cleaved by dipeptidyl peptidase IV (DPPIV) into NPY 3-36, a 34-amino acid peptide fragment. NPY 3-36, however, has drastically different properties than its parent peptide; it has been shown to have an important role in cancer symptomatology. Importantly, NPY 3-36 has angiogenic properties and, therefore, may play a role in various cancer pathways, including, but not limited to, the formation and growth of solid tumors, and metastasis.
Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA), has been used to detect NPY. ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal.
Importantly, current immunoassays for NPY measure NPY 1-36 and NPY 3-36 simultaneously without distinguishing them. In addition, they cross-react with many other breakdown products of the two main NPYs. This result is suboptimal, as the properties of NPY 1-36 and NPY 3-36 are different.
Efficient mass spectrometric techniques for the independent quantitation of NPY 1-36 and NPY 3-36 at physiologically relevant concentrations have been likewise unavailable. While matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) has been recognized as an important tool in the study of neuropeptides due to the ability directly to detect ions at a specific mass-to-charge (m/z) ratio, even in complex biological mixtures, it has thus far been limited to determining the identity of peptide fragments produced by neuropeptide-processing, -converting, and-inactivating enzymes. Nilsson, C.; et al. “Processing of Neuropeptide Y and Somatostatin in Human Cerebrospinal Fluid as Monitored by Radioimmunoassay and Mass Spectrometry,” Peptides 1998, 19(7), 1137-1146. Similarly, endogenous NPY has been qualitatively identified using a combination of chromatographic techniques and nanospray mass spectrometry. Stenfors, C.; et al. “Characterization of Endogenous Neuropeptide Y in Rat Hippocampus and Its Metabolism by Nanospray Mass Spectrometry,” J. Biol. Chem. 2997, 272(9), 5747-5751.
Therefore, there exists a need for an efficient mass spectrometric technique for the independent and simultaneous quantitation of NPY 1-36 and NPY 3-36 in a sample at physiologically relevant concentrations.